Nitrite reductase (EC 1.6.6.4) is an enzyme that catalyzes the reduction of nitrite to ammonia, a six-electron transfer reaction in which ferredoxin is the physiological electron donor. This is an important reaction in plants and is part of the nitrate assimilatory pathway in which glutamine is the end product. Between one and three isozymes of nitrite reductase appear to be present in various plants, with two such isozymes having been detected in maize. The results in maize, however, are unclear, and a better method is needed to determine the number of isozymes in particular cultivars of maize.
A nitrite reductase gene from spinach, a dicotyledenous plant (dicot), has been cloned; however, nitrite reductase genes have not yet been cloned from monocotylenous plants (monocots). Spinach, soybeans, and cotton are representative examples of dicots, while monocots include plants such as wheat, corn and rice. Monocots, including cereal grains, are of great agronomic importance and make up a large percentage of the earth's food supply. In addition, to their differing external leaf appearance, monocots and dicots are very different in their biochemistry, and in their developmental and morphological organization. Because of the significant differences between dicots and monocots, laboratory and field researchers routinely must employ different techniques and materials to study the genetic characteristics and their growth characteristics of the two groups.
Unlike nitrite reductase, nitrate reductase cDNA clones have been isolated from a variety of plants and have enabled investigators to determine the enhancing effect of the addition of nitrate on nitrate reductase activity levels in those plants (Calza et al., Mol. Gen. Genet. 209:552-562, 1987; Cheng et al., Proc. Natl. Acad. Sci. USA 83:6825-6828, 1986; and Crawford et al., Proc. Natl. Acad. Sci. USA 83:8073-8076, 1986). These papers and all other publications cited herein are hereby incorporated herein by reference.
Isolation of maize nitrite reductase complementary DNA according to this invention makes it possible to determine the effect of various environmental conditions, including the presence of nitrate, on the level of nitrite reductase mRNA of maize plants grown under the selected conditions.
It is therefore an object of this invention to provide a method to clone nitrite reductase DNA of maize.
It is a further object of this invention to provide a cloned nitrite reductase DNA that may be used to study nitrite reductase gene regulation in maize under various plant growth conditions.
It is a further object of this invention to provide methods to use a cloned nitrite reductase DNA to study the nitrate assimilation process, including nitrite reductase gene regulation, in maize.
It is a further object of this invention to provide a method to determine the number of nitrite reductase genes in maize.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.